Title: Regulation of Cross-linking of Actin Filament by IQGAP1, a Target for Cdc42
Abstract: We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1val12 (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363–23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to β- and γ-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1–216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5′-(3-O-thio)triphosphate (GTPγS)·GST-Cdc42 but not by GDP·GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTPγS·GST-Cdc42 but not GDP·GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization. We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1val12 (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363–23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to β- and γ-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1–216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5′-(3-O-thio)triphosphate (GTPγS)·GST-Cdc42 but not by GDP·GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTPγS·GST-Cdc42 but not GDP·GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization. Cdc42 and Rac1, the members of Rho small GTPases, are shown to regulate a variety of morphological events through actin cytoskeleton (for reviews see Refs. 1Mackay D. Nobes C.D. Hall A. Trends Neurosci. 1995; 18: 496-501Abstract Full Text PDF PubMed Scopus (117) Google Scholar and 2Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar). Cdc42 and Rac1 are implicated in the filopodia (3Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (883) Google Scholar, 4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar) and lamellipodia (4Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar, 5Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3076) Google Scholar) formation, respectively, in Swiss 3T3 cells. Target molecules for Cdc42 and Rac1 have thus far been identified to be serine/threonine kinase PAK (6Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1300) Google Scholar, 7Manser E. Chong C. Zhao Z.S. Leung T. Michael G. Hall C. Lim L. J. Biol. Chem. 1995; 270: 25070-25078Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (303) Google Scholar), WASP 1The abbreviations used are: WASP, Wiskott-Aldrich syndrome protein; CHD, calponin homology domain; F-actin, filamentous actin; GTPγS, guanosine 5′-(3-O-thio)triphosphate; GST, glutathione S-transferase; aa, amino acid; PAGE, polyacrylamide gel electrophoresis. (9Aspenström P. Lindberg U. Hall A. Curr. Biol. 1996; 6: 70-75Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 10Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar), and IQGAP1 (11Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 12McCallum S.J. Wu W.J. Cerione R.A. J. Biol. Chem. 1996; 271: 21732-21737Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 13Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (223) Google Scholar, 14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). PAK is activated by Cdc42 and Rac1. PAK has been proposed to be the upstream activator of c-Jun amino-terminal kinase and p38 mitogen-activated protein kinase (15Zhang S. Han J. Sells M.A. Chernoff J. Knaus U.G. Ulevitch R.J. Bokoch G.M. J. Biol. Chem. 1995; 270: 23934-23936Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). WASP is shown to be involved in the regulation of actin reorganization (10Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar, 16Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (555) Google Scholar). Despite of these intensive studies, how Cdc42 and Rac1 regulate actin cytoskeleton is still unclear. We have previously identified IQGAP1 as a target for Cdc42 and Rac1 and found that it was colocalized with the actin filament at the lamellipodia induced by insulin and Rac1val12 (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). This result led us to examine the role of IQGAP1 as a regulator of actin cytoskeleton. IQGAP1 has a so called CHD (13Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (223) Google Scholar, 17Castresana J. Saraste M. FEBS Lett. 1995; 374: 149-151Crossref PubMed Scopus (124) Google Scholar), which is also present in several actin-binding proteins, including calponin (18Winder S.J. Walsh M.P. J. Biol. Chem. 1990; 265: 10148-10155Abstract Full Text PDF PubMed Google Scholar), filamin (19Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1089-1105Crossref PubMed Scopus (433) Google Scholar), α-actinin (20Meyer R.K. Aebi U. J. Cell Biol. 1990; 110: 2013-2024Crossref PubMed Scopus (226) Google Scholar), and fimbrin (21de Arruda M.V. Watson S. Lin C.S. Leavitt J. Matsudaira P. J. Cell Biol. 1990; 111: 1069-1079Crossref PubMed Scopus (158) Google Scholar). On the other hand, filamin (22Nunnally M.H. Powell L.D. Craig S.W. J. Biol. Chem. 1981; 256: 2083-2086Abstract Full Text PDF PubMed Google Scholar), α-actinin (20Meyer R.K. Aebi U. J. Cell Biol. 1990; 110: 2013-2024Crossref PubMed Scopus (226) Google Scholar), and fimbrin (23Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6849-6853Crossref PubMed Scopus (130) Google Scholar) are known to cross-link the actin filament (24Hartwig J.H. Kwiatkowski D.J. Curr. Opin. Cell Biol. 1991; 3: 87-97Crossref PubMed Scopus (218) Google Scholar, 25Matsudaira P. Trends Biochem. Sci. 1991; 16: 87-92Abstract Full Text PDF PubMed Scopus (237) Google Scholar). It has been shown that oligomerization of filamin or α-actinin enables them to cross-link the F-actin. Cross-linking of F-actin appears to be important for the filopodia and lamellipodia formation. However, little is understood about how cross-linking of F-actin is regulated by intracellular signals. In this study, we identified actin as an IQGAP1-interacting molecule and found that IQGAP1 interacted with F-actin in vitro and cross-linked F-actin in a GTPγS·GST-Cdc42-dependent manner. We also showed here that IQGAP1 formed oligomers, suggesting that IQGAP1 cross-links the actin filament through its oligomerization. Anti-actin polyclonal antibody was kindly provided by Dr. I. Yahara (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). Anti-IQGAP1 polyclonal antibody was raised against GST-IQGAP1 (aa 1–863) as an antigen. All materials used in the nucleic acid study were purchased from Takara Shuzo Co. (Kyoto, Japan). Other materials and chemicals were obtained from commercial sources. Expression vectors pGEX2T-Cdc42 and pGEX2T-Rac1 were constructed as described previously (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Expression and purification of these GST fusion proteins were performed essentially as described (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 26Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (395) Google Scholar). To obtain GST-IQGAP1-N-1 (aa 1–863), N-2 (aa 1–216), N-3 (aa 216–683), M-1 (aa 521–914), or C-1 (aa 764–1657), cDNA fragments corresponding to the IQGAP1 fragments were subcloned into pGEX4T-1 or pGEX4T-2. Myc-IQGAP1 (aa 1–1657) and GST-IQGAP1 (aa 1–1657) were purified from overexpressingSpodoptera frugiperda insect cells. The insect cells overexpressing Myc-IQGAP1 were homogenized with Buffer A (20 mm Tris/HCl at pH 7.4, 1 mm dithiothreitol, 1 mm EDTA, and 10 μm(p-amidinophenyl)-methanesulfonyl fluoride on ice and centrifuged at 100,000 × g for 1 h at 4 °C. For cosedimentation assay, the pellets were extracted by the addition of Buffer A containing 500 mm NaCl. After shaking for 1 h at 4 °C, the extracts were centrifuged at 100,000 ×g for 1 h at 4 °C. Then Myc-IQGAP1 was purified from the supernatant by MonoQ column chromatography. For falling ball viscometry assay, GST-IQGAP1 was purified by glutathione-Sepharose column chromatography from overexpressing insect cells. For gel filtration chromatography, the insect cells overexpressing Myc-IQGAP1 were homogenized with Buffer A on ice and centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was loaded onto calmodulin-Sepharose 4B column. After washing the column with Buffer A containing 500 mm NaCl, Myc-IQGAP1 was eluted with Buffer A containing 2 m NaCl. The purity of Myc-IQGAP1 in the eluate was about 95% estimated by SDS-PAGE. The affinity purification was performed essentially as described (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 26Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (395) Google Scholar). Briefly, bovine brain cytosol was passed through glutathione beads to remove endogenous GST. Then the pass fraction was loaded on glutathione beads containing GST, GST-IQGAP1-N-1 (aa 1–863), N-2 (aa 1–216), N-3 (aa 216–683), M-1 (aa 521–914), or C-1 (aa 764–1657) as described (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar,26Amano M. Mukai H. Ono Y. Chihara K. Matsui T. Hamajima Y. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 271: 648-650Crossref PubMed Scopus (395) Google Scholar). After washing the columns, bound proteins were eluted by the addition of Buffer A containing 500 mm NaCl. F-actin was purified from an acetone powder prepared from rabbit skeletal muscle as described (27Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (982) Google Scholar). F-actin was mixed with either Myc-IQGAP1 (aa 1–1657), GST-IQGAP1-N-1 (aa 1–863), GST-IQGAP1-N-2 (aa 1–216), GST-IQGAP1-N-3 (aa 216–683), or GST-IQGAP1-M-1 (aa 521–914) and incubated at room temperature for 1 h with or without either GST, GDP·GST-Cdc42, or GTPγS·GST-Cdc42 (3 μm each) in Buffer B (20 mm Tris/HCl at pH 7.4, 0.5 mm dithiothreitol, 2 mm MgCl2, 100 mm NaCl, 1 mm EDTA, 10% (w/v) sucrose, 0.5 mm ATP, 10 μg/ml leupeptin, and 10 μm(p-amidinophenyl)-methanesulfonyl fluoride). After the incubation, 50 μl of each reaction mixture was layered onto a 100-μl sucrose barrier (20% (w/v) sucrose in Buffer B) and centrifuged at 200,000 × g for 1 h at room temperature. The supernatants and pellets were separated and subjected to SDS-PAGE (28Hughes C.A. Bennett V. J. Biol. Chem. 1995; 270: 18990-18996Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The effect of IQGAP1 on F-actin was tested using falling ball viscometry method (29MacLean-Fletcher S.D. Pollard T.D. J. Cell Biol. 1980; 85: 414-428Crossref PubMed Scopus (231) Google Scholar). GST-IQGAP1 (aa 1–1657), GST-IQGAP1-N-1 (aa 1–863), GST-IQGAP1-N-2 (aa 1–216), GST-IQGAP1-N-3 (aa 216–683), or GST-IQGAP1-M-1 (aa 521–914) was incubated at room temperature for 1 h with or without GST, GDP·GST-Cdc42, or GTPγS·GST-Cdc42 (3 μm each) in Buffer C (20 mm Tris/HCl at pH 7.4, 1 mmdithiothreitol, 7.5 mm MgCl2, 150 mm NaCl, 3.2 mm EDTA, and 0.1 mmATP). After the incubation, each sample was mixed with F-actin (3 μm) in Buffer D (20 mm Tris/HCl at pH 7.4, 1 mm dithiothreitol, 4 mm MgCl2, 140 mm NaCl, 2.5 mm EDTA, and 0.1 mmATP), and each solution was quickly sucked into a 0.1-ml micropipette. After the incubation for 1 h at room temperature, the pipettes were placed at an angle of 50 °, and the time it took a 0.5-mm steel ball to fall 2 cm was measured. Myc-IQGAP1 (25 μg) was incubated at 4 °C for 1 h with or without GDP·GST-Cdc42 or GTPγS·GST-Cdc42 (100 μg each) in Buffer E (20 mmTris/HCl at pH 7.4, 1 mm dithiothreitol, 6 mmMgCl2, 300 mm NaCl, and 1.9 mmEDTA). Superose 6 HR 10/30 (Pharmacia Biotechnology Inc.) gel filtration column was equilibrated with the Buffer A containing 200 mm NaCl. Each sample was centrifuged at 10,000 ×g for 1 h at 4 °C. The supernatant was loaded onto the column at a flow rate 0.25 ml/min, and 0.25-ml fractions were collected. An aliquot of each fraction was subjected to SDS-PAGE followed by Western blotting (30Berryman M. Gary R. Bretscher A. J. Cell Biol. 1995; 131: 1231-1242Crossref PubMed Scopus (179) Google Scholar). The amino acid sequencing of p43 protein was carried out as described (31Iwamatsu A. Yoshida-Kubomura N. J. Biochem. (Tokyo). 1996; 120: 29-34Crossref PubMed Scopus (54) Google Scholar). We and other groups have recently identified IQGAP as a target for Cdc42 and Rac1 (11Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 12McCallum S.J. Wu W.J. Cerione R.A. J. Biol. Chem. 1996; 271: 21732-21737Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 13Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (223) Google Scholar, 14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). We found that IQGAP1 accumulated at the membrane ruffling area induced by Rac1val12 and by insulin in KB cells, where cortical actin cytoskeleton was localized (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). To understand the function of IQGAP1, we attempted to identify an IQGAP1-interacting molecule(s). Bovine brain cytosol was subjected to the GST, GST-IQGAP1-N-1 (aa 1–863), or GST-IQGAP1-C-1 (aa 764–1657) coated affinity column. After washing the columns, the proteins bound to GST-IQGAP1 were eluted with 500 mm NaCl in Buffer A. Proteins with molecular masses of 43 (p43) and 41 kDa (p41) were specifically detected in the GST-IQGAP1-N-1 (aa 1–863) eluate (Fig.1 A). These proteins were not observed in either the eluate from GST or GST-IQGAP1-C-1 (aa 764–1657). Therefore, these proteins are likely to be IQGAP1-interacting molecules. To determine the molecular identity of p43, it was subjected to the amino acid sequencing. The peptide sequences derived from p43 were EITALAPSTMK and AGFAGDDAPRAVFP, both of which are identical to those of β- and γ-actin. The molecular weights of β- and γ-actin were calculated to be 41,605 and 41,661, respectively, which are almost the same as that of p43. We also confirmed that p43 was recognized by anti-actin antibody (Fig.1 B). Therefore, we concluded that p43 was actin. Identification of p41 is currently under investigation. 2Identification of p41 will be described elsewhere (study in progress). IQGAP1 has a CHD (17Castresana J. Saraste M. FEBS Lett. 1995; 374: 149-151Crossref PubMed Scopus (124) Google Scholar), which is also present in several actin-binding proteins, including calponin, filamin, α-actinin, and fimbrin, in its amino terminus (13Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (223) Google Scholar, 32Weissbach L. Settleman J. Kalady M.F. Snijders A.J. Murthy A.E. Yan Y.X. Bernards A. J. Biol. Chem. 1994; 269: 20517-20521Abstract Full Text PDF PubMed Google Scholar). To determine whether IQGAP1 directly interacts with F-actin, cosedimentation assay of recombinant IQGAP1 with F-actin was performed. Myc-IQGAP1 was cosedimentated in the presence of F-actin but not in the absence of F-actin (Fig.2 A), indicating that IQGAP1 directly interacts with F-actin. GST-IQGAP1-N-1 (aa 1–863) was also cosedimentated with F-actin, whereas IQGAP1-C-1 (aa 764–1657) was not (data not shown). We produced the indicated mutants of IQGAP1, and their interactions with F-actin were assessed. GST-IQGAP1-N-2 (aa 1–216) was cosedimentated with F-actin, whereas others were not (Fig.2 B). Therefore, the amino terminus of IQGAP1 (aa 1–216) containing the CHD is sufficient for the F-actin binding. To examine whether Cdc42 affects this binding, we used Myc-IQGAP1 (aa 1–1657) because Cdc42 does not interact with IQGAP1-N-1 (aa 1–863) but interacts with Myc-IQGAP1 (aa 1–1657) (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). The similar assay was performed in the presence or the absence of either GTPγS·GST-Cdc42 or GDP·GST-Cdc42. Neither GTPγS·GST-Cdc42 nor GDP·GST-Cdc42 affected the F-actin binding activity of Myc-IQGAP1 (aa 1–1657) (data not shown). We also examined whether IQGAP1 interacts with globular actin using a GST-IQGAP1-N-1 (aa 1–863) affinity column chromatography and found that IQGAP1 did not interact with globular actin (data not shown). Next we examined whether IQGAP1 cross-links the F-actin. Actin-based viscosity was measured by the falling ball viscometry assay (29MacLean-Fletcher S.D. Pollard T.D. J. Cell Biol. 1980; 85: 414-428Crossref PubMed Scopus (231) Google Scholar). Actin-based viscosity was markedly increased in the presence of GST-IQGAP1 (aa 1–1657) and GST-IQGAP1-N-1 (aa 1–863) in a dose-dependent manner compared with that in the absence of IQGAP1 (Fig. 3 A), suggesting that IQGAP1 cross-links F-actin. The shorter fragments, GST-IQGAP1-N-2 (aa 1–216), GST-IQGAP1-N-3 (aa 216–683), and GST-IQGAP1-M-1 (aa 521–914), did not increase the viscosity. We examined whether Cdc42 affects this activity. The similar assay was performed in the presence or the absence of GST, GTPγS·GST-Cdc42, or GDP·GST-Cdc42. GTPγS·GST-Cdc42 markedly enhanced the cross-linking activity of GST-IQGAP1 (aa 1–1657), whereas GDP·GST-Cdc42 did not (Fig. 3 B). GTPγS·GST-Cdc42 did not enhance the cross-linking activity of GST-IQGAP1-N-1 (aa 1–863) because Cdc42 did not interact with IQGAP1-N-1 (aa 1–863) (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) (data not shown). When bovine brain cytosol was subjected to the indicated mutants of IQGAP1 affinity column chromatography, endogenous IQGAP1 was specifically detected in the GST-IQGAP1-N-3 (aa 216–683) eluate (Fig.4 A). IQGAP1 was not detected in the GST, GST-IQGAP1-N-2 (aa 1–216), GST-IQGAP1-M-1 (aa 521–914), and IQGAP1-C-1 (aa 764–1657) eluates. This result suggests that IQGAP1 forms oligomers through at least the amino-terminal portion of IQGAP1 (aa 216–683). We examined whether guanine nucleotides or Cdc42 affect this interaction. A similar assay was performed in the presence or the absence of GDP, GTPγS, GDP·Cdc42, or GTPγS·Cdc42. None of these conditions affected this interaction (Fig. 4 B). Actin-cross-linking proteins, such as filamin (19Gorlin J.B. Yamin R. Egan S. Stewart M. Stossel T.P. Kwiatkowski D.J. Hartwig J.H. J. Cell Biol. 1990; 111: 1089-1105Crossref PubMed Scopus (433) Google Scholar) and α-actinin (33Suzuki A. Goll D.E. Singh I. Allen R.E. Robson R.M. Stromer M.H. J. Biol. Chem. 1976; 251: 6860-6870Abstract Full Text PDF PubMed Google Scholar), form oligomers, and the oligomerization enables them to cross-link F-actin (25Matsudaira P. Trends Biochem. Sci. 1991; 16: 87-92Abstract Full Text PDF PubMed Scopus (237) Google Scholar). Therefore, we examined whether IQGAP1 can form oligomers by gel filtration analysis. Myc-IQGAP1 appeared as broad major and minor peaks, corresponding to the molecular masses of about 300 and 500 kDa, respectively (Fig. 5). These peaks may contain monomers, dimers, and trimers, suggesting that IQGAP1 can form oligomers. GTPγS·GST-Cdc42 but not GDP·GST-Cdc42 markedly shifted the peak of IQGAP1, corresponding to the molecular mass of about 600 kDa. GTPγS·GST-Cdc42 alone was eluted as a dimer. Judging from the molecular mass of a dimer of GTPγS· GST-Cdc42 (about 100 kDa), it is likely that GTPγS·GST-Cdc42 enhances the oligomerization of IQGAP1. These results strongly suggest that the oligomerization of IQGAP1 leads to cross-linking the actin filament. We have previously found that IQGAP1 accumulated at the insulin- and Rac1val12-induced membrane ruffling area in KB cells, where cortical actin filament was observed (14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). In this study, we found that IQGAP1 directly interacts with F-actin and cross-links the actin filament. Cdc42 and Rac1 have been shown to regulate the filopodia and lamellipodia formation through the reorganization of actin filament meshwork (1Mackay D. Nobes C.D. Hall A. Trends Neurosci. 1995; 18: 496-501Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 2Chant J. Stowers L. Cell. 1995; 81: 1-4Abstract Full Text PDF PubMed Scopus (260) Google Scholar). Cross-linking of F-actin may be critical for the filopodia and lamellipodia formation. Our result strongly suggests that IQGAP1 plays an important role in these processes through the regulation of actin filament. The target molecules for Cdc42 and Rac1 have thus far been identified to be PAK (6Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1300) Google Scholar, 7Manser E. Chong C. Zhao Z.S. Leung T. Michael G. Hall C. Lim L. J. Biol. Chem. 1995; 270: 25070-25078Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 8Martin G.A. Bollag G. McCormick F. Abo A. EMBO J. 1995; 14: 1970-1978Crossref PubMed Scopus (303) Google Scholar), WASP (9Aspenström P. Lindberg U. Hall A. Curr. Biol. 1996; 6: 70-75Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 10Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar), and IQGAP1 (11Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 12McCallum S.J. Wu W.J. Cerione R.A. J. Biol. Chem. 1996; 271: 21732-21737Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 13Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (223) Google Scholar, 14Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Recently, it is reported that microinjection of activated PAK induces the filopodia formation and membrane ruffling in Swiss 3T3 cells (34Sells M.A. Knaus U.G. Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1997; 7: 202-210Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). Another group reported that expression of constitutively activated PAK induces dynamic morphological changes (35Manser E. Huang H.Y. Loo T.H. Chen X.Q. Dong J.M. Leung T. Lim L. Mol. Cell. Biol. 1997; 17: 1129-1143Crossref PubMed Google Scholar). However, these results do not exclude the possibility that other targets are involved in the filopodia and lamellipodia formation. WASP (9Aspenström P. Lindberg U. Hall A. Curr. Biol. 1996; 6: 70-75Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 10Symons M. Derry J.M.J. Karlak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar) and its isoform, N-WASP (16Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (555) Google Scholar), are shown to alter the actin filament localization and to sever the F-actin, respectively. On the basis of these observations, it is plausible that IQGAP1, together with PAK and WASP, plays an important role in the reorganization of actin filament. Actin-cross-linking proteins, such as α-actinin (20Meyer R.K. Aebi U. J. Cell Biol. 1990; 110: 2013-2024Crossref PubMed Scopus (226) Google Scholar), filamin (22Nunnally M.H. Powell L.D. Craig S.W. J. Biol. Chem. 1981; 256: 2083-2086Abstract Full Text PDF PubMed Google Scholar), and fimbrin (23Bretscher A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6849-6853Crossref PubMed Scopus (130) Google Scholar) are believed to bridge F-actin and consequently cross-link the F-actin (25Matsudaira P. Trends Biochem. Sci. 1991; 16: 87-92Abstract Full Text PDF PubMed Scopus (237) Google Scholar). Although IQGAP1 has a CHD in its amino terminus (aa 1–216) and this domain is sufficient for the interaction with F-actin, this domain lacked the F-actin-cross-linking activity. The IQGAP1-N-1 (aa 1–863) had the F-actin binding and cross-linking activities. Additionally, oligomerization of IQGAP1 is mediated by its amino-terminal portion (aa 216–683). On the basis of these observations, it is likely that the CHD is sufficient for the binding to F-actin and the portion (aa 216–683) is responsible for the oligomerization of IQGAP1. Thus, IQGAP1 can cross-link the actin filament in a similar fashion to other actin-cross-linking proteins. GTPγS·GST-Cdc42 enhanced this IQGAP1 activity when 300 nm of IQGAP1 was used, although Cdc42 did not affect the activity of IQGAP1 when more than 750 nm of IQGAP1 was used. This result suggests that Cdc42 caused a leftward shift of the curve of the IQGAP1 activity. The concentration of endogenous IQGAP1 was calculated to be about 300 nm in cultured cells, such as MTD-1A epithelial cells (data not shown). At this concentration of IQGAP1, Cdc42 can further enhance the cross-linking activity of IQGAP1. Therefore, it is plausible that IQGAP1 physiologically cross-link the actin filament in a Cdc42-dependent mannerin vivo. GTPγS·Cdc42 did not affect the interaction of IQGAP1 with F-actin, whereas GTPγS·Cdc42 enhanced the oligomerization of IQGAP1. This may account for how Cdc42 induces the cross-link of the actin filament through IQGAP1. The mechanism by which GTPγS·Cdc42 elicits the oligomerization of IQGAP1 is not known at present. A possible explanation is that GTPγS·Cdc42 affects the conformation of IQGAP1 and subsequently promotes the oligomerization of IQGAP1 through the amino-terminal portion (aa 216–683) of IQGAP1. However, this possibility may be less likely because neither GTPγS nor GTPγS·Cdc42 affected the interaction of bovine IQGAP1 with the amino-terminal portion (aa 216–683) of IQGAP1. Alternatively, Cdc42 itself may make oligomers so that IQGAP1-Cdc42 complex consequently forms additional oligomers. It has been shown that Ras and RhoA, members of small GTPases, form oligomers (36Santos E. Nebreda A.R. Bryan T. Kempner E.S. J. Biol. Chem. 1988; 263: 9853-9858Abstract Full Text PDF PubMed Google Scholar, 37Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (169) Google Scholar). If Cdc42 forms oligomers as in the case of Ras and RhoA, this possibility is likely. However, a further study to address this issue is necessary. Very recently, during the revision of this manuscript, IQGAP1 has been shown to bind F-actin and cross-link F-actin (38Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (213) Google Scholar). Our result is consistent with this observation. We showed here that GTPγS·GST-Cdc42 enhanced the F-actin-cross-linking activity of IQGAP1, possibly through enhancing the oligomerization of the IQGAP1-Cdc42 complex. Therefore, IQGAP1 appears to be a key molecule for the actin cytoskeletal reorganization regulated by Cdc42. We thank the cDNA group of Kazusa DNA Research Institute for providing a cDNA of IQGAP1, Dr. I. Yahara (Tokyo Metropolitan Institute of Medical Science, Japan) for providing an anti-actin polyclonal antibody, and Dr. E. Nishida (Kyoto University, Japan) and Dr. M. Inagaki (Aichi Cancer Center Research Institute, Japan) for helpful discussion.